Topologically engineered antibodies and Fc-fusion proteins: a new class of multifunctional therapeutic candidates for SARS-CoV-2, cancer, and other disease
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Abstract
The ability of antibodies and Fc-fusion proteins to bind multiple targets cooperatively is limited by their topology. Here we describe our discovery that ACE2 Fc-fusion proteins spontaneously cross-dimerize, forming topologically distinct “superdimers” that demonstrate extraordinary SARS-CoV-2 intra-spike cooperative binding and potently neutralize Omicron B.1.1.529 at least 100-fold better than eight clinically authorized antibodies. We also exploited cross- dimerization to topologically engineer novel superdimeric antibodies and Fc-fusion proteins with antibody-like plasma half-lives to address cancer and infectious disease therapy. These include bispecific ACE2-antibody superdimers that potently neutralize all major SARS-CoV-2 variants, and bispecific anti-cancer and anti-viral antibody superdimers that are more potent than two-antibody cocktails. Superdimers are efficiently produced from single cells, providing a new therapeutic approach to many disease indications.
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SciScore for 10.1101/2022.03.23.485397: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable A total of 24 male golden hamsters 6-8 weeks old were assigned to four groups (n=6). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources A secondary antibody, Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, F(ab’)2, Cat#109-605-097 or Affinipure Rabbit Anti-Human IgG Fcγ fragment specific, Cat#309-005-008 (Jackson ImmunoResearch, West Grove, PA), at a final concentration of 0.5 μg/mL was used to detect the unbound fraction. Anti-Human IgGsuggested: (Jackson ImmunoResearch Labs Cat# 109-605-097, RRID:AB_2337888)Biotinylated goat anti … SciScore for 10.1101/2022.03.23.485397: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable A total of 24 male golden hamsters 6-8 weeks old were assigned to four groups (n=6). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources A secondary antibody, Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, F(ab’)2, Cat#109-605-097 or Affinipure Rabbit Anti-Human IgG Fcγ fragment specific, Cat#309-005-008 (Jackson ImmunoResearch, West Grove, PA), at a final concentration of 0.5 μg/mL was used to detect the unbound fraction. Anti-Human IgGsuggested: (Jackson ImmunoResearch Labs Cat# 109-605-097, RRID:AB_2337888)Biotinylated goat anti human-Fab antibody, Cat#ab64666 (Abcam, Cambridge, UK), or biotinylated goat Anti-Human ACE-2 antibody, Cat#DY933-05 (R&D Systems, Minneapolis, MN) was used as a detection antibody. anti human-Fabsuggested: NoneAnti-Human ACE-2suggested: NoneThe Rituxan and FMC63 antibodies were labeled with AF488, Cat#ab236553, and AF647, Cat#ab269823 (Abcam), respectively, according to the manufacturer’s instructions. FMC63suggested: NoneAF647suggested: NoneExperimental Models: Cell Lines Sentences Resources Methods and Materials: Mammalian expression and purification of recombinant proteins: Recombinant proteins were expressed by transient expression in CHO-K1 cells adapted to serum- free suspension culture (TunaCHO) using a mammalian expression vector and purified by Protein A affinity chromatography (LakePharma, San Carlos, CA). CHO-K1suggested: CLS Cat# 603480/p693_CHO-K1, RRID:CVCL_0214)Live SARS-CoV-2 and NL63 neutralization assay: Infectious virus USA-WA1/2020, Cat#NR-52281, and NL63, Cat#NR-470 (BEI, Manassas, VA) was expanded using a single passage in Vero/TMPRSS2 cells. Vero/TMPRSS2suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)Experimental Models: Organisms/Strains Sentences Resources Pharmacokinetic (PK) assays: The study was conducted by The Jackson Laboratory, Bar Harbor, ME using 6-8 week old male B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ mice homozygous for the human FcRn transgene. B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJsuggested: RRID:IMSR_JAX:014565)Software and Algorithms Sentences Resources Astra v6.1.7 software (Wyatt Technology) was used for light scattering and refractive index data acquisition and processing. Astrasuggested: (ASTRA, RRID:SCR_016255)Pharmacokinetic (PK) analysis: Noncompartmental pharmacokinetic data analysis was performed using Phoenix WinNonlin v8.3 (Certara, Princeton, NJ). Phoenix WinNonlinsuggested: NoneKinetic data were obtained using IncuCyte 2021A software (Sartorius) before area-under-the-curve (AUC) (mean ± SEM) and non-linear regression analysis were performed using GraphPad Prism v9.3.1. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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